Interferon-gamma (IFN-γ) inducing factor (IGIF, IL-18) and peptide fragment thereof

ABSTRACT

A protein which induces the IFN-γ production by immunocompetent cells and has a molecular weight of 19,000±5,000 daltons on SDS-PAGE or gel filtration method and a pI of 4.8±1.0 on chromatofocusing. The protein is isolated from mouse liver and can be purified by a monoclonal antibody specific to it. The monoclonal antibody can be also used for assaying the protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.08/502,535, filed Jul. 14, 1995, issued as U.S. Pat. No. 5,912,324.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a protein which induces theinterferon-γ (hereinafter abbreviated as “IFN-γ”) production byimmunocompetent cells, and a monoclonal antibody specific to theprotein.

2. Description of the Prior Art

It is said that IFN-γ is a protein which has antiviral-, antioncotic-and immunoregulatory-activities and which is produced by immunocompetentcells stimulated with antigens and/or mitogens. Because of thesebiological activities, IFN-γ is expected to be used as an antitumoragent since the discovery, and energetically studied on clinical trialsas a therapeutic agent for malignant tumors in general including braintumors. IFN-γ preparations now commercially available are roughlyclassified into 2 groups, i.e. natural IFN-γs produced byimmunocompetent cells and recombinant IFN-γs produced by transformantsprepared by introducing DNAs which encode the natural IFN-γs intomicroorganisms of the species Escherichia coli. In such clinical trials,either of these IFN-γs is administered to patients as an “exogenousIFN-γ”.

Among these IFN-γs, the natural IFN-γ is usually produced by culturingestablished immunocompetent cells in nutrient culture media supplementedwith IFN-γ inducers to form IFN-γ, and purifying the IFN-γ. It is knownthat the type of IFN-γ inducers greatly influence on the productionyield and the facility of IFN-γ purification, as well as the safeness ofthe final products. Generally, mitogens such as concanavalin A (Con A),Lens culinaris, Phytolacca americana, endotoxin and lipopolysaccharideare used. These mitogens, however, have problems of their molecular andquality varying dependently on their origins and purification methods,as well as the difficulty of obtaining a desired amount of preparationswith a constant IFN-γ inducibility. In addition, most of these mitogensinduce unfavorable side effects when administered to living bodies, andsome of them may cause toxicity, so that it is substantially difficultto induce the IFN-γ production by the direct administration to livingbodies.

During the study of cytokines produced by mammalian cells, the presentinventors have found in mouse liver a novel substance which induces theIFN-γ production. They isolated the substance using two or moreconventional purification methods including column chromatographymainly, studied the property and feature and revealing that the realityis a protein having the following physicochemical properties:

(1) Molecular weight

19,000±5,000 daltons on gel filtration sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE);

(2) Isoelectric point (pI)

4.8±1.0 on chromatofocusing;

(3) Partial amino acid sequence

Possessing partial amino acid sequences corresponding to amino acidresidues 26-43 and 79-103 of SEQ ID NO:2; and

(4) Biological activity

Inducing the interferon-γ production by immunocompetent cells.

Such a protein with these physicochemical properties has never beenreported, and the data concludes that the protein is novel. The presentinventors energetically studied on mouse liver cells and have succeededto isolate a DNA encoding the protein. The decoding of the proteinrevealed that the DNA consists of 471 base pairs and encodes the aminoacid sequence in SEQ ID NO:3. When the DNA was introduced intomicroorganisms of the species Escherichia coli to express the productionof the present protein, the protein was produced in the culture in asatisfactorily high yield. These findings are disclosed in JapanesePatent Application No.184,162/94 applied by the present applicant.

As is described above, the present protein has an activity of inducingthe IFN-γ production by immunocompetent cells, and is expected to beused in a variety of uses as an anti-virus agent, antioncotic agent,antiseptic, immunoregulatory agent or a platelet-increasing agent.Generally, in the case of incorporating biologically active proteinsinto pharmaceuticals, the developments of methods for purifying suchproteins highly and effectively and those for assaying samplescontaining these proteins are inevitable. The material most suitable forthe purification and assay is a monoclonal antibody, but such amonoclonal antibody specific to the protein is not established.

SUMMARY OF THE INVENTION

In view of the foregoing, the object of the present invention is toprovide a novel protein which induces the IFN-γ production byimmunocompetent cells.

It is another object of the present invention to provide a DNA encodingthe protein.

It is further object of the present invention to provide a replicablerecombinant DNA which contains the DNA and a self-replicable vector.

It is yet another object of the present invention to provide atransformant obtainable by introducing the recombinant DNA into anappropriate host.

It is another object of the present invention to provide a process forpreparing the protein by using the recombinant DNA technology.

It is another object of the present invention to provide a novelmonoclonal antibody which is specific to the protein having theaforesaid physicochemical properties.

It is another object of the present invention to provide a hybridomawhich produces the monoclonal antibody.

It is another object of the present invention to provide a process forpreparing the monoclonal antibody.

It is another object of the present invention to provide a purificationmethod with the monoclonal antibody.

It is another object of the present invention to provide a method fordetecting the protein with the monoclonal antibody.

The first object of the present invention is attained by a proteinhaving the following physicochemical properties:

(1) Molecular weight

19,000±5,000 daltons on gel filtration and sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE);

(2) Isoelectric point (pI)

4.8±1.0 on chromatofocusing;

(3) Partial amino acid sequence

Possessing partial amino acid sequences corresponding to amino acidresidues 26-43 and 79-103of SEQ ID NO:2; and

(4) Biological activity

Inducing the IFN-γ production by immunocompetent cells.

The second object of the present invention is attained by a DNA whichencodes the protein.

The third object of the present invention is attained by a replicablerecombinant DNA which contains the DNA and a self-replicable vector.

The fourth object of the present invention is attained by a transformantobtainable by introducing the replicable recombinant DNA into anappropriate host.

The fifth object of the present invention is attained by a process forpreparing the protein comprising culturing the transformant in anutrient culture medium, and collecting the formed protein from theresultant culture.

The sixth object of the present invention is attained by a monoclonalantibody which is specific to a protein having the followingphysicochemical properties:

(1) Molecular weight

19,000±5,000 daltons on gel filtration and sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE);

(2) Isoelectric point (pI)

4.8±1.0 on chromatofocusing;

(3) Partial amino acid sequence

Possessing partial amino acid sequences corresponding to amino acidresidues 26-43 and 79-103 of SEQ ID NO:2; and

(4) Biological activity

Inducing the IFN-γ production by immunocompetent cells.

The seventh object of the present invention is attained by a hybridomawhich can produce the monoclonal antibody.

The eighth object of the present invention is attained by a processcomprising culturing in vivo or in vitro hybridomas capable of producingthe monoclonal antibody, and collecting the monoclonal antibody from theresultant body fluids or cultures.

The ninth object of the present invention is attained by a method forpurifying the present protein comprising contacting a monoclonalantibody specific to the protein to a mixture containing the protein andimpurities to adsorb the protein on the monoclonal antibody, anddesorbing the adsorbed protein from the monoclonal antibody.

The tenth object of the present invention is attained by a method fordetecting the present protein comprising contacting the monoclonalantibody with test samples containing the protein to immunologicallyreact them.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is an elution pattern of peptide fragments, obtained bytrypsinizing the present protein, on high-performance liquidchromatography (HPLC).

FIG. 2 is a structure of pMGTG-1, a recombinant DNA according to thepresent invention.

FIG. 3 is a structure of recombinant DNA pKGFM5.

FIG. 4 is a figure of the reactivity of the present monoclonal antibodyM-1mAb with a protein on Western blot technique.

In these figures, the symbol “MGTG-1 cDNA” means cDNA which encodes thepresent protein; “KGFM5 cDNA”, cDNA which encodes the present protein;“Ptac”, tac promoter; “GST”, glutathione S transferase gene; “AmpR”,ampicillin resistant gene; “ori”, replication initiation site ofEscherichia coli; and “rrnBT1T2”, terminator of ribosome RNA operon.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding of a novel protein whichinduces the IFN-γ production by immunocompetent cells. During studyingon cytokines produced from mammalian cells, the present inventors foundthe existence of a novel substance, which induces the IFN-γ production,in mouse liver. They isolated the substance by combining purificationmethods comprising column chromatography mainly, studied the propertyand feature and revealing that the reality is a protein having thefollowing physicochemical properties:

(1) Molecular weight

19,000±5,000 daltons on gel filtration sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE);

(2) Isoelectric point (pI)

4.8±1.0 on chromatofocusing;

(3) Partial amino acid sequence

Possessing partial amino acid sequences corresponding to amino acidresidues 26-43 and 79-103 of SEQ ID NO:2; and

(4) Biological activity

Inducing the interferon-γ production by immunocompetent cells.

The protein according to the present invention has a specific propertyof inducing the IFN-γ production when acts on immunocompetent cells.

The DNA according to the present invention expresses the production ofthe present protein by introducing the DNA into an appropriateself-replicable vector to form a recombinant DNA, and introducing therecombinant DNA into a host capable of proliferating without difficultybut inherently incapable of producing the protein.

The replicable recombinant DNA according to the present inventionexpresses the production of the present protein when introduced into ahost capable of proliferating without difficulty but inherentlyincapable of producing the protein.

The transformant produces the present protein in a desired amount with arelative easiness when cultured by the process according to the presentinvention.

The present monoclonal antibody specifically reacts with a proteinhaving specific physicochemical properties.

The present hybridoma forms the monoclonal antibody when cultured invivo and in vitro.

The present process facilitates the production of the monoclonalantibody in a desired amount.

The present purification method yields the present protein with arelatively-high purity from a mixture containing it along withimpurities.

In the present detection method, the present protein in test samplesonly exhibits an immunoreaction, so that the protein is detectedquantitatively and qualitatively by monitoring the immunoreaction with asuitable method.

The protein according to the present invention includes proteins ingeneral which have specific physicochemical properties and those derivedfrom natural sources and those prepared by the recombinant DNAtechnology. The present protein generally has a partially or totallyrevealed amino acid sequence, for example, the amino acid sequencecontaining the N-terminal in SEQ ID NO:2 and its homologous amino acidsequences. Variants, which have complementary amino acid sequences tothe one in SEQ ID NO:2, can be obtained by replacing one or more aminoacids in SEQ ID NO:2 with other amino acids without altering theinherent biological properties of the present protein. Even when usedthe same DNA and depending on hosts into which the DNA is introduced, aswell as on the components of nutrient culture media, the conditions ofcultivation temperature and pH for culturing transformants containingthe DNA, it may be formed variants, which are defective in oradditionally contain one or more amino acids near to the N-terminal inSEQ ID NO:2 while retaining the inherent biological properties of theprotein, by the modification with internal enzymes of the hosts afterthe DNA expression. The present protein includes such variants as longas they induce the IFN-γ production by immunocompetent cells.

The present protein can be prepared by culturing in nutrient culturemedia transformants with DNAs encoding the protein, and collecting theformed protein from the resultant cultures. The transformants usable inthe present invention can be obtained by introducing into appropriatehosts DNAs having the base sequence in SEQ ID NO:1, homologous basesequences to it, and complementary ones to these base sequences. One ormore bases in those base sequences can be replaced with other bases bymeans of the degeneracy of genetic code without alternating the aminoacid sequence of the present protein. To express the production of theprotein in hosts with such DNAs, one or more bases in base sequences,which encode the present protein or its variants, can be replaced withother bases.

Any DNA can be used in the present invention as long as it has one ofthose base sequences independently of their origin, i.e. those fromnatural sources or those prepared by chemical synthesis. The naturalsources include, for example, mouse liver cells from which a genecontaining the present DNA is obtainable. The preparation procedure isas follows: Remove mouse liver previously challenged with stimulantssuch as Corynebacterium parvum, BCG (Bacillus Calmette-Guérin, mitogenand lipopolysaccharide, disrupt the liver cells, and isolate the wholeDNAs from the resultant suspension. Treat the DNAs with oligo-dTcellulose or oligo-dT latex to obtain poly (A)⁺RNA, and fractionate itusing a sucrose density gradient buffer to isolate mRNA. Allow a reversetranscriptase and a polymerase to act on the mRNA as a template to formdouble-stranded cDNA, introduce the cDNA into an appropriateself-replicable vector, and introduce the resultant recombinant DNA intoan appropriate host such as Escherichia coli. Culture the resultanttransformant in a nutrient culture medium, and collect the proliferatedcells containing the DNA encoding the present protein by the colonyhybridization method. The DNA according to the present invention isobtainable by treating the transformants with conventional methods. Toartificially produce the present DNA, for example, it is prepared by thechemical synthesis based on the base sequence in SEQ ID NO:1, or byintroducing a DNA which encodes the amino acid sequence in SEQ ID NO:2into an appropriate vector to form a recombinant DNA, introducing therecombinant DNA into an appropriate host, culturing the resultanttransformant in a nutrient culture medium, isolating the proliferatedcells from the culture, and collecting plasmids containing the objectiveDNA from the cells.

The DNA was generally introduced into hosts in the form of a recombinantDNA. Such a recombinant DNA usually contains the DNA and aself-replicable vector, and it can be readily prepared by therecombinant DNA technology in general if only the DNA is in hand.Examples of such self-replicable vector are plasmid vectors such aspKK223-2, pGEX-2T, pRL-λ, pBTrp2 DNA, pUB110, YEp13, Ti plasmid, Riplasmid and pBI121. Among these vectors, pKK223-2, pGEX-2T, pRL-λ,pBTrp2 DNA, pUBilO and YEpl3 are suitably used when the present DNA isexpressed in procaryotes such as yeasts and other microorganisms of thespecies Escherichia coli and Bacillus subtilis, while Ti plasmid, Riplasmid and pBIl21 are suitably used for the expression in animal- andplant-cells.

To introduce the present DNA into these vectors, conventional methodsused in this field can be arbitrarily used: Genes containing the presentDNA and self-replicable vectors are cleaved with restriction enzymesand/or ultrasonic, and the resultant DNA fragments and vector fragmentsare ligated. To cleave genes and vectors, restriction enzymes whichspecifically act on nucleotides, more particularly, type II restrictionenzymes such as Sau 3AI, Eco RI, Hind III, Bam HI, Sal I, Xba I, Sac Iand Pst I, can be used to facilitate the ligation of DNA fragments andvector fragments. To ligate DNA fragments and vector fragments, theyare, if necessary, first annealed, then treated with a DNA ligase invivo or in vitro. The recombinant DNAs thus obtained can be readilyintroduced into appropriate hosts, and this enables the limitlessreplication of the DNAs by culturing the transformants.

The recombinant DNAs usable in the present invention can be introducedinto appropriate hosts such as yeasts and other microorganisms of thespecies Escherichia coli and Bacillus subtilis: When microorganisms ofthe species Escherichia coli are used as a host, they are cultured inthe presence of recombinant DNAs and calcium ions, and the competentcell method and the protoplast method are used when microorganisms ofthe species Bacillus subtilis are used as a host. To clone the objectivetransformants, they are selected by the colony hybridization method orby culturing all the transformants in nutrient culture media, andselecting ones which produce proteins capable of inducingimmunocompetent cells to produce IFN-γ.

The transformants thus obtained produce the present proteinintracellularly or extracellularly when cultured in nutrient culturemedia. Examples of such nutrient culture media are those in the form ofliquid in general which contain carbon sources, nitrogen sources andminerals, as well as amino acids and/or vitamins as a micronutrient. Thecarbon sources usable in the present invention include saccharides suchas starch, starch hydrolysates, glucose, fructose and sucrose. Thenitrogen sources usable in the present invention include organic andinorganic nitrogen-containing compounds such as ammonia and their salts,urea, nitrates, peptone, yeast extract, defatted soy bean, corn steepliquor, and beef extract. The transformants are inoculated into nutrientculture media and incubated at a temperature of 25-65° C. and at a pH of2-8 for about 1-10 days under aerobic conditions by theagitation-aeration method, etc., to obtain cultures containing thepresent protein. Although the cultures can be used intact as an IFN-γinducer, they are, if necessary, subjected to ultrasonication and/orcell lysis enzymes to disrupt cells, followed by filtering orcentrifuging the resultant suspensions to remove intact cells and celldebris, and further purifying the resultant supernatants containing thepresent protein. The purification methods usable in the presentinvention are, for example, those which are generally used in this fieldto purify biologically active substances, i.e. concentration, saltingout, dialysis, separatory sedimentation, gel filtration chromatography,ion-exchange chromatography, hydrophobic chromatography, affinitychromatography, chromatofocusing, gel electrophoresis, and isoelectricpoint electrophoresis, and, if necessary, two or more of them can beused in combination. The resultant purified solutions containing thepresent protein can be concentrated and/or lyophilized into liquids orsolids to meet to final uses.

As is described above, the present protein has an activity of inducingIFN-γ production by immunocompetent cells. Because of this, the presentprotein can be arbitrarily used as therapeutic and/or prophylacticagents, for example, those for virus diseases such as AIDS and condylomaacuminatum; malignant tumors such as renal cancer, granuloma, mycosisfungoides and cerebral tumor; and immune disorders such as articularrheumatism and allergy.

The present protein is allowed to coexist in nutrient culture media toinduce the IFN-γ production by immunocompetent cells, or directlyadministered to mammals for the treatment and/or prevention of IFN-γsusceptive diseases. In the former, leukocytes separated from peripheralblood of mammals, or established immunocompetent cells such as HBL-38cells, MO cells, Jurkat cells, EL-4 cells and L12-R4 cells are suspendedin nutrient culture media containing the present protein to induce theIFN-γ production. If necessary, such nutrient culture media can besupplemented with T-cell stimulants such as mitogen, interleukin 2, andanti-CD 3 antibody, and the cells are cultured at 30-40° C. and at a pHof about 5-8 for about 1-100 hours while the media were replacing withfresh ones. IFN-γ can be obtained from the resultant cultures with oneor more conventional methods in general used for purifying biologicallyactive substances, for example, concentration, salting out, dialysis,separatory sedimentation, gel filtration chromatography, ion-exchangechromatography, chromatofocusing, gel electrophoresis, and isoelectricpoint electrophoresis.

To treat and/or prevent IFN-γ susceptive diseases, the IFN-γ inducingagents according to the present invention are directly administered tomammals: For example, the agents are orally administered to mammalsafter formulated into appropriate forms, or injected to the mammalsintradermally, subcutaneously, muscularly, intravenously andperitoneally. The mammals, which can be administered with the presentprotein, are not restricted to human, and include other animals such asmouse, rat, hamster, rabbit, dog, cat, caw, horse, coat, sheep, pig andmonkey. Since the present protein has a strong IFN-γ inducibility and anextremely-low toxicity, it readily induces the IFN-γ production withonly a small amount without causing serious side effects even whenadministered to in a relatively-large amount. Thus, the present proteininduces the desired amount of IFN-γ production smoothly without strictlycontrolling the administration, so that it can be used as an IFN-γproduction inducing agent.

The present protein has a feature of strongly activating thecytotoxicity of killer cells, and, when used in combination withinterleukin 2 and/or tumor necrosis factor (TNF), it exerts a strongeffect on the therapeutic effect and/or the reduction of side effects inthe treatment of adoptive immunotherapy for malignant tumors includingsolid carcinomas such as lung cancer, renal cancer, and breast cancer.

The monoclonal antibody according to the present invention includesthose in general, which are specific to the proteins having the abovephysicochemical properties independently of their sources, origins andclasses. Examples of proteins are those which have the amino acidsequence in SEQ ID NO:3 and its homologous ones. Such homologous aminoacid sequences include those wherein one or more amino acids arereplaced with other amino acids without substantially alternating thephysicochemical properties of the protein, as well as those which one ormore amino acids are added to the N- and C-terminals in SEQ ID NO:2, andthose which are defective in one or more amino acids in the N- andC-terminals in SEQ ID NO:2.

The present monoclonal antibody can be obtained by using the protein orits antigenic fragments as an antigen. For example, the monoclonalantibody can be obtained by hybridizing antibody-producing cells,collected from mammals which had been immunized with those antigens,with cells that infinitely proliferate, cloning hybridomas capable ofproducing the present monoclonal antibody, and culturing the clones innutrient culture media in vitro.

The proteins usable as an antigen in the present invention can beobtained from mouse liver cells as disclosed in Japanese PatentApplication No.184,162/94, or obtained by culturing in nutrient culturemedia transformants into which DNAs encoding the amino acid sequence inSEQ ID NO:2 or its homologous ones are introduced. Generally, they areused in a completely-or partially-purified form. To obtain the antigenicfragments, the resultant completely- or partially-purified proteins arehydrolyzed chemically or enzymatically, or subjected to peptidesynthesis using the amino acid sequence in SEQ ID NO:2.

The immunization methods usable in the present invention includeconventional ones: For example, those which comprise injecting theantigens with or without adjuvants into mammals intravenously,subcutaneously or intraperitoneally, and feeding the mammals for aprescribed period of time. The mammals usable in the present inventionare not specifically restricted to as long as they produce the objectiveantibody-producing cells independently of their kind, size and sex.Although rodents such as rat, mouse and hamster are generally used, themost suitable mammal is chosen therefrom in view of the adaptivity tothe following mammalian cells which infinitely proliferate. Depending onthe kind and size of mammals, the total dose of antigens is generallyabout 5-500 μg/mouse and administered to in 2-5 shots at an interval ofabout 1-2 weeks. On 3-5 days after the final administration, the spleensof the mammals are extracted and dispersed in nutrient culture media toobtain a spleen cell suspension as an antibody-producing cell.

The resultant cells and mammalian cells capable of infinitelyproliferating are hybridized to obtain hybridized cells containing theobjective hybridomas. The mammalian cells capable of infinitelyproliferating generally include cell lines from mouse bone marrow suchas P3-NS1-Ag4-1 cells (ATCC TIB18), P3-X63-Ag8 cells (ATCC TIB9) andSP2/O-Ag14 cells (ATCC CRL1581), and their variants. The methods to fusecells include conventional methods wherein electric pulses and fusionaccelerators such as polyethylene glycol and sendai virus (HVJ) areused. For example, those which comprise suspending antigen-producingcells and mammalian cells capable of infinitely proliferating in a ratioof about 1:1 to 1:10 in cell fusion media containing fusionaccelerators, and incubating at about 30-40° C. for about 1-5 min. Themedia for cell fusion used in the present invention are those ingenerally used in this field such as MEM medium, RPMI 1640 medium andIscove's modified Dulbecco's medium, and it is desirable to excludeserums such as calf serum from the media.

To select the objective hybridoma, the resultant fused cells aretransferred to selection media such as HAT medium, incubated at about30-40° C. for about 3 days to 3 weeks to die cells other than theobjective hybridomas. The hybridomas are cultured in usual manner, andthe antibody secreted in the culture is tested for reactivity with thepresent protein. Conventional assays for detecting antigens such asenzyme immunoassay, radioimmunoassay and bioassay are used in thisexperiment. For example, these assays are described in detail in“Tan-kuron Kotai Jikken Manual (Monoclonal Antibody ExperimentalManual)”, edited by Sakuji TOYAMA and Tamie ANDO, pp.105-152 (1991).Hybridomas capable of producing antibodies specific to the presentprotein are speedily cloned by the limiting dilution to obtain thepresent hybridoma The monoclonal antibody according to the presentinvention can be obtained by culturing the present hydridoma in vivo orin vitro. The culture methods usable in the present invention includeconventional ones used for culturing mammalian cells. For example, inthe case of in vitro culture, the monoclonal antibody is collected fromthe resultant culture, while in the case of in vivo culture wherein thehybridoma is transplanted in warm-blooded animals and cultured in thebodies, the monoclonal antibody is collected from the animals's ascitesor blood. The later described hybridoma M-1 is characteristic of arelatively-high productivity of the monoclonal antibody and a readilyproliferation in vivo and in vitro. The methods for collecting themonoclonal antibody from the resultant cultures, ascites and bloodinclude conventional methods generally used in this field to purifyantibodies in general. For example, salting out, dialysis, filtration,concentration, centrifugation, separatory sedimentation, gel filtrationchromatography, ion-exchange chromatography, affinity chromatography,high-performance liquid chromatography (HPLC), gel electrophoresis andisoelectric point electrophoresis, can be used alone or in combination.The purified monoclonal antibody is concentrated and/or dried into aliquid or solid form to meet to final use.

The monoclonal antibody according to the present invention is extremelyuseful in the purification of the present protein on immunoaffinitychromatography. Such a purification method comprises a step ofcontacting the present monoclonal antibody with a mixture containing thepresent protein and impurities, and a step of desorbing the adsorbedprotein from the monoclonal antibody. These steps are usually carriedout in an aqueous medium. The present monoclonal antibody is generallyused under the conditions of coupling to aqueous carriers in a gel formwhich are packed in cylindrical columns, followed by feeding to thecolumns with mouse liver cell extracts, cultures of transformants, ortheir partially purified products to substantially adsorb the protein onthe monoclonal antibody. The adsorbed protein is readily desorbed bychanging the pH around the monoclonal antibody, for example, in the caseof using a monoclonal antibody belonging to IgG class, the presentprotein is eluted therefrom at an acid pH, usually, a pH of 2-3, whilein the case of using a monoclonal antibody belonging to IgM class, it iseluted at an alkaline pH, usually, a pH of 10-11.

The present purification method can purify the present protein with theminimum labor cost and time. As is described above, the present proteinhas an activity of inducing the IFN-γ production by immunocompetentcells, so that the resultant purified protein can be used as an inducerfor the IFN-γ production by cell culturing methods, and can bearbitrarily used as a therapeutic and/or prophylactic agent for viraldiseases such as AIDS and condyloma, malignant tumors such as renalcancer, granuloma, mycosis fungoides and cerebral tumor. When theprotein according to the present invention has an activity of increasingthe cytotoxicity of killer cells, it can be used in combination withinterleukin 2 and/or tumor necrosis factor to exert a remarkableefficacy on the therapeutic effect in the treatment of malignant tumorsincluding solid carcinomas such as lung cancer, renal cancer and breastcancer, and it also decreases the side effects.

The monoclonal antibody according to the present invention is widelyuseful in the field where the detection of the present protein isrequired. When the present monoclonal antibody is used in combinationwith labeled immunoassays such as radioimmunoassay, enzyme immunoassayand fluorescent immunoassay, the present protein in test samples isquickly and accurately quantified or qualified. In such analyses, thepresent monoclonal antibody is labeled with radioactive substances,enzymes and/or fluorescent substances prior to use. Since the presentmonoclonal antibody specifically binds to the present protein to causean immunoreaction, a trace amount of the present protein in test samplescan be detected by measuring the level of the immunoreaction based onthese labeled substances as a marker. As compared with bioassays,labeled immunoassays have features that it can assay many samples at thesame time in a relatively short time, low labor-cost, and high accuracy.Therefore, the present detection method is extremely useful forcontrolling the steps in the preparation of the present protein and inthe quality control of the final products. Although the presentinvention does not describe in detail the method for labeling monoclonalantibodies and the labeled assays because the present invention initself does not relate to them, examples of such are described by P.Tijssen in “Practice and Theory of Enzyme Immunoassays”, pp.196-347(1989).

The following experiments describe the present protein:

Experiment 1

Preparation of Purified Protein

To 600 8-week-old female CD-1 mice was intraperitonealy injected onemg/mouse of dead Corynebacterium parvum (ATCC 11827) which had beenobtained by preheating at 60° C. for one hour, and the mice were fed inusual manner for 7 days and intravenously injected with one μg/mouse ofa purified lipopolysaccharide derived from Escherichia coli. On 1-2hours after the intravenous injection, the mice were sacrificed bydislocating their cervical vertebrae to collect their blood from hearts,followed by removing their livers, disrupting them by a homogenizer in8-fold volumes of 50 mM phosphate buffer (pH 7.3), and extracting theresultant. The resultant extract was centrifuged at about 8,000 rpm for20 min, and an about 9 L of the resultant supernatant was admixed with asaturated ammonium sulfate in 50 mM phosphate buffer (pH 7.3) to give asaturation degree of 45 w/v %. The resultant solution was allowed tostand at 4° C. for 18 hours and centrifuged at about 8,000 rpm for 30min to obtain a 19 L supernatant containing the present protein.

The supernatant was fed to a column packed with about 4.6 L of “PHENYLSEPHAROSE”, a product of Pharmacia LKB, Uppsala Sweden, which had beenequilibrated with 50 mM phosphate buffer (pH 7.3) containing one Mammonium sulfate, and the column was washed with a fresh preparation ofthe same buffer, and fed at an SV (space velocity) 0.57 with a lineargradient of ammonium sulfate ranging from 1 M to 0.2 M in 50 mMphosphate buffer (pH 7.3). Fractions containing the present proteineluted at 0.8 M ammonium sulfate were collected and pooled into an about4.8 L solution which was then concentrated with a membrane filter,dialyzed against 20 mM phosphate buffer (pH 6.5) at 4° C. for 18 hours,and fed to a column packed with about 250 ml of “DEAE-SEPHAROSE”, aproduct of Pharmacia LKB, Uppsala, Sweden. The column was washed with afresh preparation of the same buffer and fed at an SV 1.2 with a lineargradient of sodium chloride ranging from 0 M to 0.2 M in 20 mM phosphatebuffer (pH 6.5) to elute the present protein at a concentration of about0.13 M sodium chloride.

Fractions containing the present protein were collected, pooled (about260 ml), concentrated and dialyzed against 25 mM Bis-Tris buffer (pH7.1) at 4° C. for 18 hours. The dialyzed solution was applied to acolumn packed with about 24 ml of “MONO-P”, a product of Pharmacia LKB,Uppsala, Sweden, and eluted with 10 v/v % polybuffer 74 (pH 4.0) whiledecreasing the pH from 7 to 4 to obtain an about 23 ml eluate containingthe present protein. The eluate was concentrated, fed to a column packedwith “SUPER-DEX 75”, a product of Pharmacia LKB, Uppsala, Sweden, whichhad been equilibrated with a solution containing 7 mM disodium hydrogenphosphate, 3 mM sodium dihydrogen phosphate, and 139 mM sodium chloride,and eluted with a fresh preparation of the same solution on gelfiltration chromatography to obtain fractions containing the presentprotein, eluted at fractions corresponding to about 19,000 daltons. Thefractions were pooled and concentrated for use in Experiment 2. Theyield of the present protein was about 0.6 pg/mouse.

Experiment 2

Physicochemical Property of Protein

Experiment 2-1

Molecular weight

In accordance with the method reported by U. K. Laemmli in Nature, Vol.227, pp.680-685 (1970), the purified protein prepared by the method inExperiment 1 was electrophoresed in a sodium dodecylsulfate (SDS)polyacrylamide gel free of reducing agent to mainly show a singleprotein band with an IFN-γ inducing activity at a position correspondingto about 19,000±5,000 daltons. The marker proteins used in thisexperiment were calf serum albumin (MW=67,000 daltons), ovalbumin(MW=45,000 daltons), soy bean trypsin inhibitor (MW=20,100 daltons), anda-lactalbumin (MW=14,400 daltons).

Experiment 2-2

Isoelectric Point

The purified protein in Experiment 1 was chromatofocused to give anisoelectric point of about 4.8±1.0.

Experiment 2-3

Partial Amino Acid Sequence

A portion of an aqueous solution containing the purified protein inExperiment 1 was concentrated up to a volume of about 50 μl which wasthen admixed with 25 μl of a solution containing 3 w/v % SDS, 60 v/v %glycerol, and 60 mg/ml dithiothreitol. The resultant mixture wasincubated at 50° C. for 30 min, positioned on 15 w/v % polyacrylamidegel, and electrophoresed in usual manner. The resultant gel was stainedby soaking it in a mixture solution of 10 v/v % aqueous acetic acidsolution and 50 v/v % aqueous methanol solution containing 0.1 w/v %coomassie brilliant blue R 250, destained by repeatedly washing the gelwith a mixture solution of 12 v/v % aqueous methanol solution and 7 v/v% aqueous acetic acid solution, and washed by soaking the gel indistilled water for 18 hours. A portion, which was stained with thecoomassie brilliant blue and contained the present protein, was cut outof the gel, and lyophilized.

The lyophilized gel was soaked in 0.6 ml aqueous solution consisting of100 mM sodium hydrogen carbonate containing 2 μg/ml “TPCK TRYPSIN”, 0.5mM calcium chloride, and 0.02 v/v % aqueous Tween 20 solution, followedby the incubation at 37° C. for 18 hours to trypsinize the protein. Theresultant was centrifuged to obtain a supernatant, while the resultantprecipitate was soaked in one ml of one v/v % aqueous trifluoroacetatecontaining 0.001 v/v % Tween 20, shook for 4 hours at ambienttemperature, and centrifuged to obtain a supernatant. The newly formedprecipitate was successively treated similarly as above with 70 v/vaqueous trifluoroacetate containing 0.001 v/v Tween 20 and with 50 v/v %aqueous acetonitrile to obtain a supernatant. The resultant supernatantand the above supernatant already obtained in the above were pooled andconcentrated up to 250 μl, and the concentrate was centrifugallyfiltered.

The resultant aqueous solution containing peptide fragments was fed to“HPLC ODS-120T”, a column for HPLC commercialized by Tosoh Corporation,Tokyo, Japan, which had been previously equilibrated with 0.1 v/vaqueous trifluoroacetate, and the column was washed with 0.1 v/v %aqueous trifluoro acetate, and fed with 0.1 v/v % trifluoro acetate at aflow rate of 0.5 ml/min while the concentration of aqueous acetonitrilewas increasing from 0 v/v % to 70 v/v % and the concentration of peptidein the eluate was monitoring by a spectrophotometer at wave lengths of214 nm and 280 nm. Fractions eluted about 75 min and about 55 min afterthe initiation of the elution were respectively collected (hereinafternamed “peptide fragment A” and “peptide fragment B”). The elutionpattern was in FIG. 1.

The peptide fragments A and B were analyzed on “MODEL 473 A”, a proteinsequencer commercialized by Perkin-Elmer Corp., Instrument Div.,Norwalk, USA, and revealing that they have the amino acid sequencescorresponding to amino acid residues 26-43 and 79-103 of SEQ ID NO:2.

Experiment 2-4

Biological Activity

Experiment 2-4(a)

Induction of the IFN-γ Production by Immunocompetent Cell

BDF1 Female mouse spleen, 8-week-old, was extracted and dispersed inserum-free RPMI 1640 medium (pH 7.4), and the cells were washed with afresh preparation of the same medium, and soaked in Gei buffer (pH 8.0)to hemolyze. The resultant spleen cells were suspended in RPMI 1640medium (pH 7.4) supplemented with 10 v/v % calf serum to give a celldensity of 1×10⁷ cells/ml, fed to a cell-separatory nylon wool columncommercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan, andincubated in an incubator at 37° C. for an hour under 5 v/v % C02conditions. Thereafter, T-cells were collected from the column byfeeding to the column with RPMI 1640 medium (pH 7.4) supplemented with10 v/v % calf serum, and washed with a fresh preparation of the samebuffer. The resultant cells were used in the following experiment forIFN-γ induction.

0.15 ml aliquots of a mouse T-cell suspension in RPMI 1640 medium (pH7.4) with a cell density of 1×10⁷ cells/ml were injected into 96-wellmicroplates, and to each well was added a present purified protein,which had been diluted with 0.05 ml RPMI 1640 medium (pH 7.4)supplemented with 10 v/v % calf serum albumin. The cells in themicroplates were incubated in the presence or in the absence of 0.5μg/ml concanavalin A in an incubator at 37° C. for 24 hours under 5 v/v% CO₂ conditions. From each well 0.1 ml of the culture supernatant wascollected and assayed for IFN-γ production level by conventional enzymeimmunoassay (EIA). As a control, a sample free of the present purifiedprotein was provided and treated similarly as above. The standard mouseIFN-γ preparation Gg02-901-533, obtained from The National Institutes ofHealth, USA, was used as an IFN-γ standard in this experiment, and theactivity was expressed in terms of international units (IU).

As a result, no significant IFN-γ production was found with the controlsample but found with the test sample: The present protein induced about2-2,000 IU IFN-γ and about 2-200 IU IFN-γ from 1×10⁶ mouse T-cells whenthe T-cells were respectively incubated with and without 0.02-10 μg/mlof concanavalin A. The results confirm that the present protein has anactivity of inducing the IFN-γ induction by immunocompetent cells.

Throughout the present specification, one unit activity of the presentprotein is defined as an amount of which induces 160 IU IFN-γ productionwhen assayed in the presence of concanavalin A (0.5μg/ml).

Experiment 2-4(b)

Augmentation of Cytotoxicity of Killer Cell

Similarly as in Experiment 2-4(a) mouse spleen cells were suspended inRPMI 1640 medium (pH 7.2) containing 100 μg/ml kanamycin, 5×10⁻⁵ M2-mercaptoethanol, and 10 v/v % calf serum to give a cell density of1×10⁷ cells/ml. The cell suspension was mixed with 0, 1, 5 or 10units/ml of a recombinant human interleukin 2, placed in a 25-ml cultureflask, admixed with 0, 0.8, 4, 20 or 100 units/ml of the purifiedprotein, and incubated in an incubator at 37° C. for 72 hours under 5v/v % CO₂ conditions. Thereafter, the resultant cells were washed with afresh preparation of the same RPMI 1640 medium (pH 7.2), and suspendedtogether with YAC-1 cells (ATCC TIB160), which were previously labeledwith radioactive sodium chromate, to give a cell ratio of 20/1 or 40/1(effective cells/target cells) in a fresh preparation of the same RPMI1640 medium (pH 7.2). The cell suspension was poured in 96-wellmicroplates and incubated in an incubator at 37° C. for 4 hours under 5v/v % C0₂ conditions, followed by determining the radioactivity of ⁵¹Crin the resultant supernatant by a γ-ray counter. The results were inTable 1.

The results in Table 1 show that the present protein has an activity ofinducing the cytotoxicity of killer cells, and the activity is augmentedby interleukin 2.

TABLE 1 Cytotoxicity (%) Factor Ratio The present protein Interleukin 2(Effective cells/Target cells) (unit/ml) (unit/ml) 40/1 20/1 100 0 48.646.0 20 0 35.5 27.5 4 0 33.0 17.7 0.8 0 22.9 14.5 0 0 0.1 0.0 100 1 55.855.2 20 1 54.2 46.4 4 1 40.5 26.4 0.8 1 22.1 10.3 0 1 0.4 0.0 100 5 63.659.1 20 5 62.2 49.1 4 5 56.2 44.6 0.8 5 38.4 23.4 0 5 1.0 0.2 100 1067.8 56.5 20 10 67.7 59.9 4 10 62.8 54.1 0.8 10 46.2 31.7 0 10 1.0 0.5

No protein having the above identified physicochemical properties hasbeen known, and this confirms that it is a novel protein. The presentinventors isolated mRNA from mouse liver cells, collected a DNA fragmentwhich partially encodes the present protein by the reversetranscription-polymerase chain reaction (RT-PCR) in the presence of aprimer which was chemically synthesized by using the mRNA as a templatebased on the partial amino acid sequence revealed in Experiment 2-3, andenergetically studied a cDNA library, prepared from the mRNA by usingthe DNA fragment as a probe, to obtain a DNA fragment in SEQ ID NO:1which contains the 5′-terminus and consists of 471 base pairs. Thedecoding of the base sequence revealed that the present protein containsan amino acid sequence in SEQ ID NO:2 which consists of 157 amino acidsand contains the N-terminal. In SEQ ID NO:2 the symbol “Xaa” as an aminoacid means “Met (methionine)” or “Thr (threonine)”.

The sequential techniques used to reveal the amino acid sequence andbase sequence in SEQ ID NOs:2 and 1 are summarized in the below:

(1) The present protein is isolated from mouse liver cells and highlypurified by combining conventional purification methods comprisingchromatography as a main technique;

(2) The resultant purified protein was digested with trypsin, and 2polypeptide fragments were isolated from the resultant mixture anddetermined for amino acid sequence;

(3) From mouse liver cells, mRNA was collected, and a DNA fragment whichpartially encodes the present protein was prepared by the reversetranscription-polymerase chain reaction (RT-PCR) in the presence of aprimer which was chemically synthesized by using the mRNA as a templatebased on the partial amino acid sequences revealed in the above. The DNAfragments were screened by using an oligonucleotide as a probe which hadbeen chemically synthesized based on these partial amino acid sequences,followed by collecting a DNA fragment which partially encodes thepresent protein;

(4) A cDNA library was prepared with the mRNA as a template andhybridized with the DNA fragment as a probe, followed by collecting atransformant which strongly hybridized with the DNA fragment;

and

(5) A cDNA was isolated from the transformant, and the base sequence wasdetermined and decoded. The comparison of the decoded amino acidsequence and the partial amino acid sequence revealed that the basesequence encodes the present protein.

The following Experiment 3 is to explain the above techniques (3) to(5), and the techniques in themselves used therein are commonly known inthe art, for example, those disclosed by J. Sumbrook et al. in“Molecular Cloning, A Laboratory Manual”, 2nd edition (1989), publishedby Cold Spring Harbor Laboratory Press, New York, USA, and by MasamiMURAMATSU in “Rabo-Manual for Genetic Technology” (1988), published byMaruzen Co., Ltd., Tokyo, Japan.

Experiment 3

Base Sequence of DNA and Amino Acid Sequence of Protein

Experiment 3-1

Preparation of Whole RNA

Three g of wet mouse liver cells, similarly prepared by the method inExperiment 1, was weighed, soaked in 20 ml of a mixture solutioncontaining 6 M guanidine isothiocyanate, 10 mM sodium citrate, and 0.5w/v SDS, and disrupted with a homogenizer. 35-ml centrifugation tubeswere injected with 25 ml of 0.1 M EDTA (pH 7.5) containing 5.7 M cesiumchloride, and 10 ml of the homogenized cells were overlaid on the upperpart of the solutions in the tubes, followed by centrifuging the tubesat 25,000 rpm for 20 hours to collect RNA fractions. The fractions werepooled, distributed into 15-ml centrifugation tubes, and mixed withequal volumes of a mixture solution of chloroform and isobutanol (=4:1by volume). The tubes were vibrated for 5 min and centrifuged at 4° C.and at 10,000 rpm for 10 min, and the formed water layers werecollected, pooled, mixed with 2.5-fold volumes of ethanol, and allowedto stand at −20° C. for 2 hours to precipitate the whole RNAs. Theprecipitate was collected, pooled, washed with 75 v/v % aqueous ethanolsolution, and dissolved in 0.5 ml of sterilized distilled-water for usein the following experiment. The yield of the RNAs was about 4 mg, on adry solid basis (d.s.b.).

Experiment 3-2

Preparation of DNA Fragments Encoding Partially the Present Protein

One μg of the whole RNAs in Experiment 3-1 was mixed with 4 μl of 25 mMmagnesium chloride, 2 μl of a solution of 10×PCR buffer consisting of100 mM Tris-HCl buffer (pH 8.3) and 500 mM potassium chloride, 8 μl ofone mM dNTP mix, one μl of a solution containing one unit/μl RNaseinhibitor, one μl of a solution containing 2.5 units/μl reversetranscriptase, and one μl of 2.5 μM random hexamer, and further mixedwith sterilized distilled-water to give a total volume of 20 μl. Themixture solution was placed in 0.5 ml reaction tubes, and, in usualmanner, successively incubated at 25° C. for 10 min, at 42° C. for 30min, at 99° C. for 5 min, and at 5° C. for 5 min to effect the reversetranscriptase reaction, followed by recovering an aqueous solutioncontaining the first strand cDNA.

To 20 μl of the aqueous solution were added 4 p1 of 25 mM magnesiumchloride, 8 μl of 10×PCR buffer, 0.5 μl of a solution containing 2.5units/μl of AmpliTaq DNA polymerase commercialized by Perkin-ElmerCorp., Instrument Div., Norwalk, USA, and one pmole of primer 1 or 2 asa sense primer or an anti-sense primer. The mixture solution was volumedup to 100 μl with sterilize d distilled-water, and, in usual manner,successively incubated at 94° C. for one min, at 45° C. for 2 min, andat 72° C. for 3 min in a cyclic manner for 40 cycles to amplify a DNAfragment, which partially encodes the present protein, by using thefirst strand cDNA as a template. The primers 1 and 2 areoligonucleotides, which were chemically synthesized based on the aminoacid sequences of Pro-Glu-Asn-Ile-Asp-Asp-Ile (residues 88-94 SEQ ID NO:2) and Phe-Glu-Asp-Met-Thr-Asp-Ile (residues 29-35 SEQ ID NO: 2) in SEQID NOs:1 and 2, and they have base sequences of5-ATRTCRTCDATRTTYTCNGG-3′ (SEQ ID NO: 3) and 5′-TTYGARGAYATGACNGAYAT-3 ′(SEQ ID NO: 4), respectively.

A portion of the resultant PCR product was fractionated onelectrophoresis in 2 w/v % agarose gel, transferred onto a nylon film,fixed with 0.4 N sodium hydroxide, washed with 2×SSC, air-dried, soakedin a prehybridization solution containing 5×SSPE, 5×Denhard's solution,0.5 w/v e SDS and 100 pg/ml of denatured salmon sperm DNA, and incubatedat 65° C. for 3 hours. An oligonucleotide as a probe 1 having a basesequence of 5′-TTYGARGARATGGAYCC-3′ (SEQ ID NO: 5) was synthesized basedon the amino acid sequence of Phe-Glu-Glu-Met-Asp-Pro corresponding toamino acid residues 82-87 of SEQ ID NO:2, and labeled with [γ-³²P]ATPand T4 polynucleotide kinase. The nylon film was soaked in a solutioncontaining one pmole of the probe 1, 5×SSPE, 5×Denhardt's solution, 0.5w/v % SDS, and 100 μg/ml of a denatured salmon sperm DNA, and incubatedat 45° C. for 24 hours to effect hybridization. The resultant nylon filmwas washed with 6×SSC and autoradiographed in usual manner and revealingthat the PCR product contained the objective DNA fragment.

The remaining PCR product was mixed with “pT7 BLUE T”, a plasmid vectorcommercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, an adequateamount of T4 ligase, and further mixed with 100 mM ATP up to give aconcentration of one mM, followed by the incubation at 16° C. for 18hours to insert the DNA fragment into the plasmid vector. Therecombinant DNA thus obtained was introduced into Escherichia coli NoVaBlue strain, a microorganism of the species Escherichia colicommercialized by Pharmacia LKB, Uppsala, Sweden, to obtain atransformant which was then inoculated into a medium plate containing 10g/l bactotryptone, 2.5 g/l sodium chloride, 15 g/l bacto-agar, 100 mg/lampicillin, 40 mg/l X-Gal and 23.8 mg/lisopropyl-p-β-thiogalacto-pyranoside (hereinafter abbreviated as“IPTG”), and incubated at 37° C. for 24 hours to form colonies. A nylonfilm was in usual manner positioned on a medium plate and allowed tostand for about 30 seconds to attach the colonies thereunto. The nylonfilm was then detached from the medium plate and soaked for 7 min in asolution containing 0.5 N sodium hydroxide and 1.5 M sodium chloride toeffect cell lysis. Thereafter, the nylon film was soaked for 3 min in1.5 M sodium chloride in 0.5 M Tris-HCl buffer (pH 7.2), washed with2×SSC, soaked in 0.4 N sodium hydroxide for 20 min to fix the DNA,washed with 5×SSC, air-dried, soaked in a prehybridization solutioncontaining 5×SSPE, 5×Denhardt's solution, 0.5 w/v % SDS, and 100 μg/mldenatured salmon sperm DNA, and incubated at 65° C. for 3 hours. Thecolonies on the nylon film were in usual manner hybridized with theprobe 1, washed with 6×SSC, and autoradiographed similarly as above,followed by selecting from the medium plate transformants which stronglyhybridized with the probe 1.

The transformants were inoculated in L-broth (pH 7.2) containing 100pg/ml ampicillin and incubated at 37° C. for 18 hours, followed bycollecting cells from the culture and collecting recombinant DNA byconventional SDS-alkali method. The analysis of the dideoxy methodrevealed that the recombinant DNA contained a DNA fragment consisting ofbase sequences which correspond to those at positions from 85 to 281 inSEQ ID NO:1.

Experiment 3-3

Preparation of mRNA

0.05 ml of an aqueous solution containing the whole RNAs in Experiment3-1 was placed in a test tube, admixed with 0.5 ml of 10 mM Tris-HClbuffer (pH 7.5) containing one mM EDTA and 0.1 w/v % SDS, and volumed upto one ml with sterilized distilled-water. To the mixture was added oneml “OLIGOTEX-dT30 SUPER”, an oligo-d(T)₃₀ latex commercialized by NipponRoche K.K., Tokyo, Japan, followed by the incubation at 65° C. for 5 minto denature the RNAs and the cooling for 3 min in an ice-chilled bath.The resultant mixture was admixed with 0.2 ml of 5 M sodium chloride,incubated at 37° C. for 10 min, and centrifuged at 10,000 rpm at 25° C.for 10 min. The precipitate in the form of a pellet was suspended in 0.5ml sterilized distilled-water, and incubated at 65° C. for 5 min toextract mRNA from the oligo-d(T)₃₀ latex. The yield of the mRNA wasabout 5 μg.

Experiment 3-4

Preparation of cDNA Library

cDNA Library was prepared from the mRNA in Experiment 3-3 by using “cDNASYNTHESIZING SYSTEM PLUS”, a cDNA cloning kit commercialized by AmershamCorp., Div., Amersham International, Arlington Heights, USA. Theprocedures were as follows: To 1.5-ml reaction tube were successivelyadded 4 μl of a solution for synthesizing the first strand cDNA, one μlsodium pyrophosphate solution, one μl of a solution of human placentaribonuclease inhibitor, 2 μl deoxynucleotide triphosphate mix, and oneμl oligo-dT primer. The resultant mixture was mixed with 2 μl of mRNA inExperiment 3-3, volumed up to 19 μl with sterilized distilled-water,mixed with one μl of a solution containing 20 units of reversetranscriptase, and incubated at 42° C. for 40 min to obtain a reactionmixture containing the first strand cDNA.

The mixture thus obtained was mixed with 37.5 μl of a solution forsynthesizing the second strand cDNA, 0.8 units of ribonuclease H derivedfrom Escherichia coli, and 23 units of DNA polymerase, and volumed up to100 μl with sterilized distilled-water. The resultant mixture wassuccessively incubated at 12 C for 60 min and at 22° C. for 60 min,mixed with 2 units of T4 DNA polymerase, and incubated at 37° C. for 10min to obtain a reaction mixture containing the second strand cDNA. Tothe reaction mixture was added 4 μl of 0.25 M EDTA (pH 8.0) to suspendthe reaction, and the resultant was in usual manner extracted withphenol and chloroform and treated with ethanol to precipitate theobjective cDNA, followed by recovering the precipitate.

To the cDNA thus obtained were added 2 μl L/K buffer, 250 μmole Eco RIadaptor, and 2.5 units of T4 DNA ligase in this order, and the resultantsolution was volumed up to 20 μl with sterilized distilled-water, andincubated at 15° C. for 16 hours to ligate the Eco RI adaptor to theboth ends of the cDNA. The reaction mixture was mixed with 2 μl of 0.25M EDTA to inactivate the remaining enzyme, and subjected to molecularsieve chromatography to remove intact Eco RI adaptor. To the resultantwere added 40 μl L/K buffer and 80 units of T4 polynucleotide kinase,and the mixture was volumed up to 400 μl with sterilizeddistilled-water, followed by the incubation at 37° C. for 30 min tomethylate the Eco RI cleavage sites. The resultant mixture was extractedwith phenol and chloroform and treated with ethanol to precipitate theobjective DNA, followed by recovering the DNA. To the DNA were added 1.5μl of L/K buffer containing an adequate amount of λgt 10 arms, and 2.5units of T4 DNA ligase, and the resultant solution was volumed up to 15μl with sterilized distilled-water, incubated at 15° C. for 16 hours toeffect ligation, and subjected to conventional in vitro packaging methodto obtain a phage containing a recombinant λDNA.

Experiment 3-5

Cloning of Recombinant DNA

A seed culture of Escherichia coli NM514 strain was in usual mannerinfected with the phage in Experiment 3-4, and the infected cells wereinoculated in an agar plate (pH 7.0) containing 10 g/l bactotrypton, 5g/l bacto-yeast extract, 10 g/l sodium chloride and 15 g/l bacto-agar,and incubated at 37° C. for 6 hours to form plaques. The agar plate wascovered with a nylon film and allowed to stand for about 30 seconds toattach the plaques thereunto. The nylon film was detached from theplate, and successively soaked in an aqueous solution containing 0.5 Msodium hydroxide and 1.5 M sodium chloride for 2 min and in 0.5 MTris-HCl buffer (pH 7.0) containing 1.5 M sodium chloride for 5 min. Thenylon film was washed with 5×SSC, air-dried, soaked in a solutioncontaining 5×SSPE, 5×Denhardt's solution, 0.5 w/v % SDS, and 100 μg/mldenatured salmon sperm DNA, and incubated at 65° C. for 3 hours.Thereafter, the resultant nylon film was incubated in a solutioncontaining an adequate amount of DNA fragment as a probe 2 obtained inExperiment 3-2 and labeled with ³²p by “READY PRIME DNA LABELLINGSYSTEM”, a DNA labeling kit commercialized by Amersham Corp., Div.,Amersham International, Arlington Heights, USA, 5×SSPE, 5×Denhardt'ssolution, 0.5 w/v % SDS, and 100 pg/ml of denatured salmon sperm DNA,and the mixture was incubated at 60° C. for 20 hours to effecthybridization. The resultant was subjected to autoradiography similarlyas above to select phage DNA clones which strongly hybridized with theprobe 2.

With conventional techniques, the clones were amplified in Escherichiacoli, followed by the extraction of a recombinant DNA from the cells.The recombinant DNA was cleaved with Eco RI, a restriction enzyme.Plasmid vector pUCl9 (ATCC 37254) was cleaved with the same restrictionenzyme, and the resultant cleaved DNA fragments and plasmid fragmentswere ligated with DNA ligase to obtain a recombinant DNA which was thenintroduced into Escherichia coli JM109 (ATCC 53323) by conventionalcompetent cell method to obtain a transformant.

Experiment 3-6

Determination of Base Sequence and Amino Acid Sequence

The transformant in Experiment 3-5 was inoculated into L-broth (pH 7.2)and cultured at 37° C. for 18 hours under shaking conditions. Theresultant proliferated cells were collected and treated withconventional SDS-alkali method to obtain a recombinant DNA containingthe DNA according to the present invention. The analysis on an automaticsequencer using a fluorophotometer revealed that the recombinant DNAcontains the base sequence from the 5′-terminus in SEQ ID NO:1. Thedecoding of the base sequence indicated that it encodes the amino acidsequence containing the N-terminal in SEQ ID NO:1. The amino acidsequence contains the partial amino acid sequences in corresponding tothose at positions from 79 to 103 and from 26 to 43 in SEQ ID NO:2, andthis means that the present protein contains the amino acid sequencecontaining the N-terminal in SEQ ID NO:2, and that it is encoded by aDNA containing the base sequence from the 5′-terminus in SEQ ID NO:1.

As is described above, the present inventors have found the presentprotein, which induces IFN-γ production by immunocompetent cells,through their long term research. Unlike conventional proteins, thepresent protein has specific physicochemical properties. The presentinvention is to provide the protein by applying the recombinant DNAtechnology.

The preparation of the present protein using the transformants will beexplained in detail with reference to the following Examples:

EXAMPLE 1

Replicable Recombinant DNA and Transformant

The first strand cDNA was prepared from the whole RNAs in Experiment 3-1by using “GeneAmp RNA PCR Kit”, a PCR kit commercialized by Takara ShuzoCo., Ltd., Tokyo, Japan. The procedures were as follows: To a 0.5-mlreaction tube were added 4 μl of 25 mM magnesium chloride, 2 μl of10×PCR buffer, 8 μl of one mM dNTP mix, one μl of one unit/μl RNaseinhibitor, one μl of 2.5 units/μl of reverse transcriptase, one μl of2.5 μM random hexamer, and one μl of the whole RNAs in Experiment 3-1,and the mixture was volumed up to 20 μl with sterilized distilled-water.The resultant mixture was successively incubated at 25° C. for 10 min,at 42° C. for 30 min, at 99° C. for 5 min, and at 5° C. for 5 min toobtain a reaction mixture containing the first strand cDNA.

Twenty μl of the reaction mixture was mixed with 4 μl of 25 mM magnesiumchloride, 8 μl of 10×PCR buffer, 0.5 μl of 2.5 units/μl of AmpliTaq DNApolymerase, and adequate amounts of sense primer and anti-sense primeras shown by the base sequences of 5′-CGAGGGATCGAACTTTGGCCGACTTC-3′ (SEQID NO: 6) and 5′-CGAGGAATTCCTAACTTTGATGTAAG-3′ (SEQ ID NO: 7) which werechemically synthesized based on the amino acid sequences near to the N-and C-terminals in SEQ ID NO:2, and the resultant mixture was volumed upto 100 μl with sterilized distilled-water. The mixture was in usualmanner successively incubated at 94° C. for one min, at 55° C. for 2min, and at 72° C. for 3 min, and the successive incubation was repeated40 cycles. The resultant PCR product was cleaved with Bam HI and Eco RIas a restriction enzyme to obtain a Bam HI-Echo RI DNA fragment.

To an adequate amount of sterilized distilled-water were added 100 ng ofthe fragment, 10 ng of “pGEX-2T”, a plasmid vector commercialized byPharmacia LKB, Uppsala, Sweden, which had been cleaved with Bam HI andEcho RI as a restriction enzyme, an adequate amount of T4 DNA ligase,and 10 mM ATP in an amount of which gives the final concentration of onemM, followed by incubating the mixture solution at 16° C. for 18 hours.The recombinant DNA thus obtained was introduced into Escherichia coliDH5 strain (ATCC 53868) to obtain a transformant which was theninoculated into L-broth (pH 7.2) containing 50 μg/l of ampicillin,followed by the incubation at 37° C. for 18 hours and extracting theobjective recombinant DNA by conventional SDS-alkali method.

The recombinant DNA was named “pMGTG-1” and analyzed for structure onthe dideoxy chain termination method and revealing that, as is shown inFIG. 2, in pMGTG-1, MGTG cDNA which has the base sequence of SEQ ID NO:1is positioned in the downstream of the Tac promotor and the gene forglutathione S transferase.

EXAMPLE 2

Preparation of Protein by Transformant

A transformant obtained by the method in Example 1 was inoculated inL-broth (pH 7.2) containing 50 μg/ml of ampicillin, and cultured at 37°C. for 18 hours under shaking conditions. One v/v % of the proliferatedtransformants as a seed was inoculated into 18 L of a fresh preparationof the same medium, and cultured at 37° C. under aeration-agitationconditions until the absorbance at a wavelength of 650 nm (A₆₅₀) of theculture reached to about 0.6, followed by adding IPTG to the culture togive a concentration of one mM. Thereafter, the resultant culture wasincubated for 5 hours and centrifuged to separate cells which were thensuspended in a mixture solution (pH 7.3) containing 150 mM sodiumchloride, 16 mM disodium hydrogen phosphate, and 4 mM sodium dihydrogenphosphate, treated in usual manner with ultrasonication, and centrifugedto remove cell debris to obtain a supernatant.

The supernatant was fed to a column packed with “GLUTATHIONE SEPHAROSE4B”, a gel commercialized by Pharmacia LKB, Uppsala, Sweden, which hadbeen equilibrated with 50 mM Tris-HCl buffer (pH 7.5) supplemented with150 mM sodium chloride, and the column was washed with a freshpreparation of the same buffer and fed with 50 mM Tris-HCl buffer (pH8.0) supplemented with 5 mM reducing glutathione to elute proteins.Fractions containing proteins were pooled, mixed with calcium chlorideto give a concentration of 2.5 mM together with 1,000 units of thrombin,and incubated at 25° C. for 18 hours. The reaction mixture was fed to acolumn packed with “GLUTATHIONE SPEPHAROSE 4B”, which had beenequilibrated with 50 mM Tris-HCl buffer (pH 7.5) supplemented with 150mM sodium chloride, followed by recovering non-adsorbed fractions.Thereafter, the fractions were pooled, concentrated, lyophilized toobtain a solid preparation containing the present protein with aspecific activity of about 5×10⁵ units/mg protein in a yield of about 3mg per one L of the culture.

Similarly as in Experiment 2, the purified protein was studied on thephysicochemical properties and revealing that it has a molecular weightof 19,000±5,000 daltons on gel filtration and SDS-PAGE, and a pI of4.8±1.0 on chromatofocusing. The testing by the method in Experiment 2-4revealed that the purified protein effectively induces the IFN-γproduction by immunocompetent cells independently of the presence ofconcanavalin A (Con A), and strongly augments the cytotoxicity of killercells. This is an evidence that the present protein can be prepared bythe recombinant DNA technology.

EXAMPLE 3

Preparation of Hybridoma M-1

EXAMPLE 3-1

Preparation of transformant KGFM5

To 0.5-ml reaction tube were added 8 μl of 25 mM magnesium chloride, 10μl of 10×PCR buffer, one μl of 25 mM dNTP mix, one μl of 2.5 units/μlAmpliTaq DNA polymerase, one ng of a recombinant DNA containing a DNAencoding the present protein and having the base sequence in SEQ ID NO:1which is prepared from a phage DNA clone according to the methoddescribed in Japanese Patent Application No.184,162/94, and an adequateamount of a sense primer and an anti-sense primer comprising the basesequences represented by 5′-GAGGAATTCTGGAGGAAGGTACCATGAACTTTGGCCGACTTC-3′ (SEQ ID NO: 8) and 5′-GCGAAAGCTTCTAACTTTGATGTAAG-3′ (SEQ IDNO: 9). The resultant mixture was volumed up to 100 μl with sterilizeddistilled-water, and successively incubated at 94° C. for one min, at43° C. for one min, 72° C. for one min, at 60 for one min, and at 70° C.for one min. The successive incubation was repeated 37 cycles to effectpolymerase chain reaction (PCR) to make restriction sites of EcO RI andHind III at the 5′- and 3′-termini respectively.

Ten ng of the resultant PCR product, “pCR-Script SK(+)” commercializedby Stratagene Cloning Systems, California, USA, was in usual mannerligated with DNA ligase to obtain a recombinant DNA which was thentransformed by introducing it into “XL-1 Blue MRF′Kan”, a microorganismof the species Escherichia coli commercialized by Stratagene CloningSystems, California, USA. The transformant was inoculated into L-broth(pH 7.2) containing 50 μg/ml of ampicillin, and cultured at 37° C. for18 hours under shaking conditions. The resultant culture was centrifugedto obtain transformants, and from which a recombinant DNA was isolatedby applying conventional SDS-alkali method. A portion of the recombinantDNA was provided and analyzed on the dideoxy method and revealing thatthe recombinant DNA was coupled with the DNA having the base sequence inSEQ ID NO:1, and the material DNA was correctly amplified by the PCR.

The remaining recombinant DNAs were in usual manner cleaved with Eco RIand Hind III as a restriction enzyme. By using “DNA Ligation Kit Version2”, a DNA ligation kit commercialized by Takara Shuzo Co., Ltd., Tokyo,Japan, 0.1 μg of the resultant Eco RI-Hind III DNA fragment and 10 ng of“pKK223-3”, a plasmid vector commercialized by Pharmacia LKB, Uppsala,Sweden, which had been previously cleaved with the above restrictionenzymes, were ligated by reacting them at 16° C. for 30 min to obtain“pKGFM5”, a replicable recombinant DNA. With competent cell method,Escherichia coli Y1090 strain (ATCC 37197) was transformed by therecombinant DNA pKGFM5, and the formed transformant “KGFM5” wasinoculated into L-broth (pH 7.2) containing 50 μg/ml ampicillin, andcultured at 37° C. for 18 hours under shaking conditions. The resultantculture was centrifuged to collect transformants, and a portion of whichwas treated with the SDS-alkali method to extract the objectiverecombinant DNA pKGFM5. The analysis on the dideoxy method revealed thatKGFM5 cDNA having the base sequence in SEQ ID NO:1 was coupled to thesite of the downstream of the Tac promotor.

EXAMPLE 3-2

Production of Protein by Transformant FGFM5

L-Broth (pH 7.2) containing 50 μg/ml was sterilized by an autoclave andcooled to 37° C. which was then inoculated with transformant KGFM5 inExample 3-1, and incubated at the same temperature for 18 hours undershaking conditions to obtain a seed culture. Eighteen L of a freshpreparation of the same L-broth was placed in a 20-L jar fermenter,sterilized similarly as above, and cooled to 37° C., which was theninoculated with one v/v % of the seed culture, and incubated at the sametemperature for 8 hours under aeration-agitation conditions. Theresultant culture was centrifuged to obtain cells which were thensuspended in a mixture solution (pH 7.3) containing 150 mM sodiumchloride and 16 mM disodium hydrogen phosphate, and 4 mM sodiumdihydrogen phosphate, disrupted by ultrasonic and centrifuged to removecell debris, followed by recovering the resultant supernatant.

Ammonium sulfate was added to and completely dissolved in thesupernatant up to give a concentration of 40 w/v % under ice-chillingconditions, allowed to stand, and centrifuged to obtain a supernatant.The supernatant thus obtained was mixed with ammonium sulfate up to givea concentration of 85 w/v %, stirred at 4° C. for 25 hours andcentrifuged to obtain a sediment containing the present protein whichwas then dissolved in 150 mM phosphate buffer (pH 6.6) containing 1.5 Mammonium sulfate. The resultant solution was fed to a column packed with“PHENYL SEPHAROSE”, a product commercialized by Pharmacia LKB, Uppsala,Sweden, which had been previously equilibrated with 10 mM phosphatebuffer (pH 6.6) containing 1.5 M ammonium sulfate. The column was washedwith a fresh preparation of the same buffer, and fed with a lineargradient ranging from 1.5 M to 0 M ammonium sulfate in 10 mM phosphatebuffer (pH 6.6).

Fractions eluted at about 0.9 M ammonium sulfate were pooled andconcentrated with a membrane filter, and the concentrate was dialyzedagainst 10 mM phosphate buffer (pH 6.5) at 4° C. for 18 hours. Thedialyzed solution was fed to a column packed with “DEAE 5PW”, a productcommercialized by Tosoh Corporation, Tokyo, Japan, which had beenpreviously equilibrated with 10 mM phosphate buffer (pH 6.5), and thecolumn was washed with a fresh preparation of the same buffer and fedwith a linear gradient ranging from 0 M to 0.2 M in 10 mM phosphatebuffer (pH 6.5), followed by collecting fractions eluted at about 0.1 Msodium chloride.

The fractions were pooled and concentrated, and the concentrate thusobtained was fed to a column packed with “SUPER-DEX 75”, a product ofPharmacia LKB Uppsala, Sweden, which had been previously equilibratedwith phosphate buffer and fed with a fresh preparation of the samephosphate buffer to elute fractions corresponding to a molecular weightof about 19,000 daltons. Thus, an aqueous solution containing about 4.7mg of a purified protein. The total yield of the protein in thispurification steps was about 26%.

The analysis according to the method as disclosed in Japanese PatentApplication No.184,162/94 revealed that the purified protein has thefollowing physicochemical properties.

The analysis of the purified protein on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under non-reducingconditions revealed that it showed a main protein band which correspondsto a molecular weight of 19,000±5,000 daltons and has an activity ofinducing IFN-γ, and had an isoelectric point of 4.8±1.0 onchromatofocusing. Two peptide fragments obtained after trypsinizationhad amino acid sequences in corresponding to amino acid residues 26-43and 79-103 of SEQ ID NO:2, respectively.

EXAMPLE 3-3

Preparation of hybridoma M-1

A purified protein obtained by the method in Example 3-2 and completeFreund adjuvant were intraperitoneally injected to 10-week-old SD ratsat a dose of 20 μg/rat. Thereafter, the rats were injected 2 times withthe same dose at an interval of 2 weeks, and intravenously injected withthe same dose one week after the final injection. On 3 days after theintravenous injection, the spleens were extracted and dispersed toobtain a cell suspension.

The spleen cells and SP2/0-Ag14 cells (ATCC CRL 1581) derived from mousemyeloma were suspended in serum-free RPMI 1640 (pH 7.2) to give celldensities of 3×10⁴ cells/ml and 1×10⁴ cells/ml, respectively, and thecells suspension was centrifuged to collect the resultant precipitate.One ml of serum-free RPMI 1640 medium (pH 7.2) containing 50 w/v %polyethylene glycol with an average molecular weight of 1,500 daltonswas added to the precipitate drop by drop over one min, and thesuspension was incubated at 37° C. for one min, and to which was addeddrop by drop serum-free RPMI 1640 medium (pH 7.2) up to give a totalvolume of 50 ml. The mixture was centrifuged to collect sediment whichwas then suspended in HAT medium. The suspension was distributed into96-well microplate in an amount of 200 μl/well, and incubated at 37° C.for one week, followed by selecting hybridomas. The reactivity between apurified protein obtained by the method in Example 3-2 and the antibodysecreted in the culture supernatant in each well was studied on enzymeimmunoassay, followed by selecting hybridomas which formed antibody thatreacted with the purified protein. According to conventional manner, thehybridoma was repeatedly subjected to the limited dilution to obtainhybridoma M-1 which forms the present monocional antibody.

EXAMPLE 4

Preparation of Monoclonal Antibody M-1mAb and Analysis on Western BlotTechnique

EXAMPLE 4-1

Preparation of Monoclonal Antibody M-1mAb

Hybridoma M-1 obtained by the method in Example 3-3 was suspended inRPMI 1640 medium (pH 7.2) supplemented with 5 v/v e calf serum to give acell density of about 1×10⁶ cells/ml, and incubated at 37° C. under 5v/v % CO₂ conditions while the culture was stepwisely scaling up. Whenthe cell density reached to the prescribed level, 1×10⁷ cells/rabbit ofhybridoma M-1 was intraperitoneally injected into 5-week-old hamsters,which had been immunosuppressed with an anti-hamster thymus antibodyprepared from rabbits and intraperitoneally injected with 0.5 ml/hamsterof pristane, and fed for one week in usual manner.

The hamsters were collected their ascites which were then diluted withphosphate buffer by 3 times, mixed with ammonium sulfate to give asaturation degree of 50 w/v %, allowed to stand at 4° C. for 24 hours,and centrifuged. The resultant sediment was collected, dialyzed against20 mM potassium dihydrogen phosphate (pH 6.7) at 4° C. overnight, andthe dialyzed solution was fed to a column packed with hydroxyapatitewhich had been previously equilibrated with a fresh preparation of thesame buffer. The column was fed with a linear gradient ranging from 20mM to 300 mM in an aqueous potassium dihydrogen phosphate solution (pH6.7) to obtain an aqueous solution containing the present monoclonalantibody M-1mAb. The yield was about 5 mg/hamster. Conventional analysisof the monoclonal antibody revealed that it is a class of IgM.

EXAMPLE 4-2

Analysis on Western blot technique

A mixture solution, consisting of 100 mg dithiothreitol, 0.5 ml of 10w/v % aqueous SDS solution, one ml of glycerol, was mixed with apurified protein obtained by the method in Example 3-2, and theresultant mixture was incubated at 37° C. for one hour and subjected toSDS-PAGE. The resultant gel was in usual manner transferred to anitrocellulose membrane which was then soaked in a culture supernatantof hybridoma M-1 for one hour, washed with 50 mM Tris-HCl buffer (pH7.5) containing 0.05 v/v % tween 20 to remove excessive amount ofantibody. The resultant nitrocellulose membrane was soaked for one hourin phosphate buffer containing an anti-rat immunoglobulin antibody whichwas prepared from rabbits and labeled with horseradish peroxidase,washed with 50 mM Tris-HCl buffer (pH 7.5) containing 0.05 v/v % tween20, and soaked in 50 mM Tris-HCl buffer (pH 7.5) containing 0.005 v/v %hydrogen peroxide and 0.3 mg/ml diamino benzine to effect coloration.

As a control, a system, where a purified protein derived from mouseliver cell obtained by the method in Example 6 or a recombinant humaninterleukin 12 is used in place of the purified protein, is provided andtreated similarly as above. The marker proteins used in this experimentwere calf serum albumin (MW=67,000 daltons), ovalbumin (MW=45,000daltons), carbonic anhydrolase (MW=30,000 daltons), trypsin inhibitor(MW=20,100 daltons) and α-lactalbumin (MW=14,400 daltons).

As is shown in FIG. 4, the monoclonal antibody M-1mAb specificallyreacted with the purified protein obtained by the method in Example 3-2(lane 1), and the purified protein obtained by the method in Example 6(lane 2). This indicates that the present monoclonal antibodyspecifically reacts with proteins having specific physicochemicalproperties independently of their preparations.

EXAMPLE 5

Purification of Protein on Immunoaffinity Chromatography

EXAMPLE 5-1

Preparation of Gel for Immunoaffinity Chromatography

Eighty mg of monoclonal antibody M-1mAb obtained by the method inExample 4-1 was weighed and dialyzed against 0.1 M borate buffer (pH8.5) containing 0.5 M sodium chloride at 4° C. overnight. Four g of“CNBr-activated sepharose 4B”, a product of Pharmacia LKB, Uppsala,Sweden, as a water-soluble carrier, was swelled in one mM aqueouschloric acid solution, and washed with a fresh preparation of the sameaqueous solution and 0.1 M borate buffer (pH 8.5) containing 0.5 Msodium chloride in this order, admixed with about 10 ml of the abovedialyzed aqueous solution of monoclonal antibody, and successivelygently stirred at ambient temperature for 2 hours and at 4° C.overnight. The formed gel was first washed with one M aqueous ethanolamine solution (pH 8.0), then successively washed with 0.1 M boratebuffer (pH 8.5) containing 0.5 M sodium chloride, and 0.1 M acetatebuffer (pH 4.0) containing 0.5 M sodium chloride, and these washingsteps were repeated 5 cycles. Finally, the resultant gel was washed withphosphate buffer to obtain a gel for immunoaffinity chromatography.Conventional analysis revealed that about 6 mg monoclonal antibodyM-1mAb bound to one ml gel.

EXAMPLE 5-2

Purification of Protein on Immunoaffinity Chromatography

Ten ml of the gel for immunoaffinity chromatography in Example 5-1 wasinjected into a plastic cylindrical column, and the column was washedwith phosphate buffer and loaded with 10 ml of fractions eluted fromphenyl sepharose column containing about 0.1 mg/ml of the presentpurified protein obtained by the method in Example 3-2. Thereafter, thecolumn was washed with a fresh phosphate buffer and fed with 35 mMaqueous ethylamine solution (pH 10.8), followed by recovering fractionswith IFN-γ inducing activity. The fractions were pooled, concentrated,and measured for IFN-γ inducing activity and protein content andrevealing that a purified protein with a purity of at least 95% wasobtained in a yield of nearly 100% to the material.

EXAMPLE 6

Purification of Protein on Immunoaffinity Chromatography

Six hundred 8-week-old female mice were intraperitoneally injected withone mg/mouse of dead cells of Corynebacterium parvum strain (ATCC 11827)prepared by heating at 60° C. for one hour, fed in usual manner for 7days, and intravenously injected with one μg/mouse of a purifiedlipopolysaccharide derived from Escherichia coli. One to 2 hours afterthe intravenous injection, the mice were sacrificed, collected bloodform their hearts, extracted their livers, and disrupted with ahomogenizer in 8-fold volumes of 50 mM phosphate buffer (pH 7.3) toeffect extraction. The resultant extract was centrifuged at about 8,000rpm for 20 min to obtain an about 9 L supernatant which was then mixedwith 50 mM phosphate buffer (pH 7.3) containing saturated ammoniumsulfate up to give a saturation degree of 45 w/v %, allowed to stand at4° C. for 18 hours, and centrifuged at about 8,000 rpm for 30 min toobtain an about 19 L supernatant containing the present protein.

The supernatant thus obtained was fed to a column packed with about 4.6L of “PHENYL SEPHAROSE”, a product of Pharmacia LKB, Uppsala, Sweden,which had been previously equilibrated with 50 mM phosphate buffer (pH7.3) containing one M ammonium sulfate, and the column was washed with afresh preparation of the same buffer, and fed with a linear gradient ofammonium sulfate ranging from 1 M to 0.2 M in 50 mM phosphate buffer (pH7.3) at an SV (space velocity) 0.57. About 4.8 L of fractions containingthe present protein eluted at a concentration of about 0.8 M ammoniumsulfate, was concentrated with a membrane, dialyzed against 20 mMphosphate buffer (pH 6.5) at 4° C. for 18 hours, and fed to a columnpacked with about 250 ml of “DEAE-SEPHAROSE”, a product of PharmaciaLKB, Uppsala, Sweden. Thereafter, the column was washed with a freshpreparation of the same buffer and fed with 20 mM phosphate buffer (pH6.5) containing sodium chloride ranging from 0 M to 0.2 M at an SV 1.2to elute the present protein at about 0.13 M sodium chloride.

An about 260 ml eluate containing the present protein was collected,concentrated and purified similarly as in Example 5 to obtain a purifiedprotein with a purity of at least 95% in a yield of nearly 100% to thematerial.

EXAMPLE 7

Detection of Protein on Enzyme Immunoassay

According to conventional method, rabbits were immunized with a purifiedprotein obtained by the method in Example 3-2, and the rabbits werecollected their blood, and from which IgG antibody was isolated,dissolved in phosphate buffer to give 20 pg/ml. One hundred μl aliquotsof the resultant solution were distributed into 96-well microplateswhich were then incubated at ambient temperature for 3 hours, followedby removing supernatants containing IgG in the wells, adding to thewells 100 μl/well of phosphate buffer containing one w/v % calf serumalbumin, and allowing the microplates to stand at 4° C. overnight.

From the microplates supernatants were removed, and the microplates werewashed with phosphate buffer containing 0.05 v/v % tween 20. A purifiedprotein obtained by the method in Example 3-2 was diluted with phosphatebuffer containing 0.5 w/v % calf serum albumin to give a prescribedconcentration, and the diluted solution was added to the wells in avolume of 100 μl/well, followed by the reaction at ambient temperaturefor 2 hours under shaking conditions. The wells were washed withphosphate buffer containing 0.05 v/v % tween 20, mixed with 100 μl/wellof monoclonal antibody M-1mAb labeled with biotin, followed by thereaction at ambient temperature for 2 hours under shaking conditions.Thereafter, the wells were washed with phosphate buffer containing 0.05v/v % tween 20, and 100 μl/well of a complex of horseradish peroxidaseand streptoavidin, and subjected to immunological reaction at ambienttemperature for 2 hours under shaking conditions. Thereafter, the wellswere washed with phosphate buffer containing 0.05 v/v % tween 20, andthe activity of horseradish peroxidase coupled to the purified proteinwas measured based on the absorbance at a wavelength of 492 nm. Theresults were in Table 2.

TABLE 2 Absorbance at wave- Concentration of length of 492 nm Relativeerror protein (pg/ml) (mean value of triplicate) (%) 2,000 1.490 ± 0.0302.0 1,000 0.723 ± 0.007 1.0 500 0.370 ± 0.020 5.4 250 0.210 ± 0.010 4.8100 0.090 ± 0.010 11.1 50 0.054 ± 0.004 6.7 0 0.017 ± 0.003 17.6

As is evident from Table 2, the present detection method can accuratelydetect at least about 50-2,000 μg/ml of the present protein.

EXAMPLE 8

Detection of Protein on Radioimmunoassay

According to conventional method, rabbits were immunized with a purifiedprotein obtained by the method in Example 3-2 and collected their blood,and from which IgG antibody was isolated. The antibody thus obtained wasin usual manner allowed to adsorb on polystyrene beads forradioimmunoassay, and the resultant beads were allowed to stand at 4° C.overnight to obtain an immobilized antibody.

The immobilized antibody, a bead with the antibody, was placed in a testtube, mixed with 0.2 ml of a purified protein, obtained by the method inExample 3-2, which had been diluted with phosphate buffer containing 0.5w/v % calf serum albumin, and allowed to stand at 4° C. for 4 hours.Thereafter, the resultant bead was washed with phosphate buffercontaining 0.05 v/v % tween 20 and 0.5 w/v % calf serum albumin,contacted with 0.2 ml (1×10⁵ cpm) of monoclonal antibody M-1mAb,obtained by the method in Example 4-1 which had been previously labeledwith ¹²⁵I, and allowed to stand at 4° C. overnight. From the system, anexcessive amount of labeled antibody was removed, and the resultant beadwas washed with phosphate buffer containing 0.05 v/v % tween 20 and 0.5w/v % calf serum albumin, followed by counting the radioactivity of thebead. The results were in Table 3.

TABLE 3 Concentration of Count (cpm) Relative protein (pg/ml) (meanvalue of triplicate) error (%) 1,150 15,900 ± 700  4.4 575 9,100 ± 2002.2 288 4,700 ± 200 4.3 144 2,300 ± 50  2.2 72 1,217 ± 4  0.3 0  159 ±4  2.6

As is evident from Table 3, the detection method can accurately detectat least about 100-1,200 μg/ml of the protein.

In summary the present invention is based on the finding of a novelprotein which induces the IFN-γ production by immunocompetent cells. Thepresent protein is generally a substance with a partially or totallyrevealed amino acid sequence which has a stable activity of inducingIFN-γ production by immunocompetent cells. Therefore, the presentprotein is widely used as an IFN-γ inducer for the IFN-γ production bythe cell culture method and as a therapeutic and/or prophylactic agentin general for IFN-γ susceptive diseases such as viral diseases,malignant tumors and immunopathies.

The present protein has a strong IFN-γ inducibility so that it caninduce the desired amount of IFN-γ production with only a relativelysmall amount. The protein dose not cause serious side effects even whenadministered to in a relatively large amount because of its extremelylow toxicity. Therefore, the present protein has an advantage that itquickly induces the desired amount of IFN-γ production without strictlycontrolling the dose. The present protein has an outstanding activity ofincreasing the cytotoxicity of killer cells and inducing a strongactivity on the therapeutic effect and/or the reduction of side effectsin the treatment of adoptive immunotherapy for malignant tumorsincluding solid carcinomas such as lung cancer, renal cancer and breastcancer.

The present protein with these useful properties can be obtained in adesired amount by using the present DNA encoding the protein.

The present invention is a significant invention that exerts such aremarkable effect and gives a great contribution to this field.

While there has been described what is at present considered to be thepreferred embodiments of the invention, it will be understood thatvarious modifications may be made therein, and it is intended to coverin the appended claims all such modifications as fall within the truespirit and scope of the invention.

9 471 base pairs nucleic acid single linear cDNA not provided CDS 1..471/note= Xaa in position 70 is Met or Thr 1 AAC TTT GGC CGA CTT CAC TGTACA ACC GCA GTA ATA CGG AAT ATA AAT 48 Asn Phe Gly Arg Leu His Cys ThrThr Ala Val Ile Arg Asn Ile Asn 1 5 10 15 GAC CAA GTT CTC TTC GTT GACAAA AGA CAG CCT GTG TTC GAG GAT ATG 96 Asp Gln Val Leu Phe Val Asp LysArg Gln Pro Val Phe Glu Asp Met 20 25 30 ACT GAT ATT GAT CAA AGT GCC AGTGAA CCC CAG ACC AGA CTG ATA ATA 144 Thr Asp Ile Asp Gln Ser Ala Ser GluPro Gln Thr Arg Leu Ile Ile 35 40 45 TAC ATG TAC AAA GAC AGT GAA GTA AGAGGA CTG GCT GTG ACC CTC TCT 192 Tyr Met Tyr Lys Asp Ser Glu Val Arg GlyLeu Ala Val Thr Leu Ser 50 55 60 GTG AAG GAT AGT AAA AYG TCT ACC CTC TCCTGT AAG AAC AAG ATC ATT 240 Val Lys Asp Ser Lys Xaa Ser Thr Leu Ser CysLys Asn Lys Ile Ile 65 70 75 80 TCC TTT GAG GAA ATG GAT CCA CCT GAA AATATT GAT GAT ATA CAA AGT 288 Ser Phe Glu Glu Met Asp Pro Pro Glu Asn IleAsp Asp Ile Gln Ser 85 90 95 GAT CTC ATA TTC TTT CAG AAA CGT GTT CCA GGACAC AAC AAG ATG GAG 336 Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly HisAsn Lys Met Glu 100 105 110 TTT GAA TCT TCA CTG TAT GAA GGA CAC TTT CTTGCT TGC CAA AAG GAA 384 Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu AlaCys Gln Lys Glu 115 120 125 GAT GAT GCT TTC AAA CTC ATT CTG AAA AAA AAGGAT GAA AAT GGG GAT 432 Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys AspGlu Asn Gly Asp 130 135 140 AAA TCT GTA ATG TTC ACT CTC ACT AAC TTA CATCAA AGT 471 Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser 145 150155 157 amino acids amino acid linear protein not provided 2 Asn Phe GlyArg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn 1 5 10 15 Asp GlnVal Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met 20 25 30 Thr AspIle Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile 35 40 45 Tyr MetTyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser 50 55 60 Val LysAsp Ser Lys Xaa Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile 65 70 75 80 SerPhe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser 85 90 95 AspLeu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu 100 105 110Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu 115 120125 Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp 130135 140 Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser 145 150 15520 base pairs nucleic acid single linear other nucleic acid /desc =“Oligonucleotide” not provided 3 ATRTCRTCDA TRTTYTCNGG 20 20 base pairsnucleic acid single linear other nucleic acid /desc = “Oligonucleotide”not provided 4 TTYGARGAYA TGACNGAYAT 20 17 base pairs nucleic acidsingle linear other nucleic acid /desc = “Oligonucleotide” not provided5 TTYGARGARA TGGAYCC 17 26 base pairs nucleic acid single linear cDNAnot provided 6 CGAGGGATCG AACTTTGGCC GACTTC 26 26 base pairs nucleicacid single linear cDNA not provided 7 CGAGGAATTC CTAACTTTGA TGTAAG 2642 base pairs nucleic acid single linear cDNA not provided 8 GAGGAATTCTGGAGGAAGGT ACCATGAACT TTGGCCGACT TC 42 26 base pairs nucleic acid singlelinear cDNA not provided 9 GCGAAAGCTT CTAACTTTGA TGTAAG 26

What is claimed is:
 1. An isolated peptide fragment obtainable from apolypeptide of SEQ ID NO:2, where Xaa at residue 70 is methionine orthreonine, and capable of being used as an antigen to which a monoclonalantibody is prepared, wherein said polypeptide of SEQ ID NO:2 is capableof inducing IFN-γ production by immunocompetent cells and has amolecular weight of about 19,000±5,000 daltons on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and an isoelectric pointof about 4.8±1.0 on chromatofocusing.
 2. The isolated peptide fragmentaccording to claim 1, which consists of an amino acid sequencecorresponding to residues 26 to 43 or residues 79 to 103 of SEQ ID NO:2.3. An isolated collection of peptide fragments obtainable from apolypeptide of SEQ ID NO:2, where Xaa at residue 70 is methionine orthreonine, and capable of being used as antigens to which monoclonalantibodies are prepared, wherein said polypeptide of SEQ ID NO:2 iscapable of inducing IFN-γ production by immunocompetent cells and havinga molecular weight of about 19,000±5,000 daltons on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and an isoelectricpoint of about 4.8±1.0 on chromatofocusing.
 4. The isolated collectionof peptide fragments according to claim 3, wherein two of said peptidefragments of the collection consists of amino acid sequencescorresponding to residues 26 to 43 and residues 79 to 103 of SEQ IDNO:2.
 5. The isolated collection of peptide fragments according to claim3, which are generated by trypsin cleavage.
 6. A purified proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2, where Xaa at residue 70 of SEQ ID NO:2 is methionine orthreonine, and a variant of SEQ ID NO:2 in which one or more amino acidresidues are deleted from the N-terminus or one amino acid residue isreplaced with a different amino acid residue, but which retains thecapability of the protein comprising SEQ ID NO:2 to induce IFN-γproduction by immunocompetent cells.
 7. The purified protein accordingto claim 6, wherein said amino acid sequence consists of SEQ ID NO:2,where Xaa at residue 70 of SEQ ID NO:2 is methionine or threonine. 8.The purified protein according to claim 6, wherein said amino acidsequence consists of SEQ ID NO:2 in which one or more amino acidresidues are deleted from the N-terminus or one amino acid residue isreplaced with a different amino acid residue, but which retains thecapability of the protein comprising SEQ ID NO:2 to induce IFN-γproduction by immunocompetent cells.
 9. The purified protein accordingto claim 6, wherein the protein has the following physico-chemicalproperties: (1) Molecular weight 19,000±5,000 daltons on gel filtrationand sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE); (2) Isoelectric point (pI) 4.8±1.0 on chromatofocusing; (3)Partial amino acid sequence Possessing the partial amino acid sequenceof SEQ ID NOs:1 and 2; and (4) Biological activity Inducing interferon-γproduction by immunocompetent cells.